Heme biosynthetic enzymes are essential for life and have been conserved throughout evolution. They are associated with human diseases and are attractive targets for the development of antibiotics, fungicides, and herbicides. In an effort to understand mechanisms of action we are determining protein structures and performing associated mutagenic and biochemical analyses. The current proposal emphasizes determination of structures of enzymes in complex with substrate/intermediate/product. We are focusing on U3S (4th enzyme of the heme biosynthesis pathway), URO-D (5th enzyme), and CPO (6th enzyme). We have developed novel methods for crystallization of these enzymes with their highly oxygen sensitive substrates and products. Preliminary structures are available for URO-D with substrate and product, and for U3S with product. We also have a preliminary structure of CPO in complex with its oxidized substrate, which is a known inhibitor of the enzyme. These structures are guiding mutagenesis and biochemical studies aimed at advancing understanding of mechanism. For example, we have constructed a single chain version of the URO-D dimer that can be used to test the proposal that the two continguous active sites interact functionally. The synthesis of heme and the other tetrapyrrole cofactors requires insertion of a central metal ion. Despite similarities in tetrapyrrole and metal substrates, the reactions are catalyzed by a diverse set of non- homologous chelatase enzymes. We aim to determine the structure of CobN, the catalytic subunit of a heterotrimeric cobaltochelatase, a class for which structural data are not currently available. We also aim to determine a variety of ligand complex structures for representatives of all three tetrapyrrole chelatase structural classes. This effort will apply the methods developed initially for our study of U3S, URO-D, and CPO.